Novel prostate-associated kallikrein

ABSTRACT

The present invention provides a human prostate-associated kallikrein (HPAK) and polynucleotides which identify and encode HPAK. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding HPAK and a method for producing HPAK. The invention also provides for antibodies or antagonists specifically binding HPAK, and their use, in the prevention and treatment of diseases associated with expression of HPAK. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding HPAK for the treatment of diseases associated with the expression of HPAK. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding HPAK.

FIELD OF THE INVENTION

[0001] This invention relates to nucleic acid and amino acid sequencesof a novel prostate-associated kallikrein and to the use of thesesequences in the diagnosis, prevention, and treatment of cancer anddisorders of the prostate.

BACKGROUND OF THE INVENTION

[0002] Prostate-specific antigen (PSA) is a 33 kD glycoproteinsynthesized in the epithelial cells of the prostate gland. It is ansecreted serine protease of the kallikrein family. PSA has been shown todigest the seminal vesicle protein, semenogelin, parathyroidhormone-related protein, and insulin-like growth factor-bindingprotein-3 (Henttu P. et al. (1994) Ann. Med. 26: 157-164; Cramer S. D.et al. (1996) J. Urol. 156: 526-531).

[0003] Genes encoding the three human kallikreins, tissue kallikrein(KLK1), glandular kallikrein (KLK2), and APS are located in a cluster atchromosome map position 19q 13.2-q13.4 (Riegmen P. H. (1992) Genomics14: 6-11). PSA shares more extensive homology with KLK2 than with KLK1.Both PSA and KLK2 are produced by prostate epithelial cells and theirexpression is regulated by androgens. Three amino acid residues werefound to be critical for serine protease activity, residues H₆₅, D₁₂₀,and S₂₁₃ in PSA (Bridon D. P. et al. (1995) Urology 45: 801-806).Substrate specificity, described as chymotrypsinogen-like (with KLK2) ortrypsin-like (with PSA) is thought to be determined by S₂₀₇ in PSA andD₂₀₉ in KLK2 (Bridon et al., supra). KLK1 is chymotrypsinogen-like andexpressed in the pancreas, urinary system, and sublingual gland. KLK1,like the other kallikreins, is made as a pre-pro-protein and isprocessed into an active form of 238 amino acids by cleavage of a 24amino acid terminal signal sequence (Fukushima D. et al. (1985)Biochemistry 24: 8037-8043).

[0004] Adenocarcinoma of the prostate accounts for a significant numberof malignancies in men over 50, with over 122,000 new cases occurringper year, in the United States alone. Prostate-specific antigen (PSA) isthe most sensitive marker available for monitoring cancer progressionand response to therapy. Serum PSA is elevated in up to 92% of patientswith prostatic carcinoma, depending upon tumor volume. Since PSA is alsomoderately elevated in patients with benign prostate hyperplasia,additional techniques are needed to distinguish between the two.

[0005] Discovery of proteins related to PSA and the polynucleotidesencoding them satisfies a need in the art by providing new compositionsuseful in diagnosis, prevention, and treatment of cancer and disordersof the prostate.

SUMMARY OF THE INVENTION

[0006] The present invention features a novel prostate-associatedkallikrein hereinafter designated HPAK and characterized as havingchemical and structural similarity to human pancreatic kellikrein andother kallikreins.

[0007] Accordingly, the invention features a substantially purified HPAKwhich has the amino acid sequence shown in SEQ ID NO:1.

[0008] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode HPAK. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0009] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0010] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode HPAK. Thepresent invention also features antibodies which bind specifically toHPAK, and pharmaceutical compositions comprising substantially purifiedHPAK. The invention also features the use of agonists and antagonists ofHPAK. The invention also features a method for producing HPAK using thehost cell, and methods for treating cancer and prostatic hyperplasia byadministering an antagonist to HPAK.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of HPAK. The alignment was producedusing MacDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd.,San Bruno, Calif.).

[0012]FIG. 2 shows the amino acid sequence alignments among HPAK (SEQ IDNO:1), human pancreatic kallikrein (GI 186653; SEQ ID NO:3), and Africanrat renal kallikrein (GI 55527; SEQ ID NO:4). The alignment was producedusing the multisequence alignment program of DNASTAR™ software (DNASTARInc, Madison Wis.).

[0013]FIG. 3 shows the hydrophobicity plot (MacDNASIS PRO software) forHPAK, SEQ ID NO: 1; the positive X axis reflects amino acid position,and the negative Y axis, hydrophobicity.

[0014]FIG. 4 shows the hydrophobicity plot for human pancreatickallikrein, SEQ ID NO:3.

[0015]FIG. 5 shows the northern analysis for SEQ ID NO:2. The northernanalysis was produced electronically using LIFESEQ™ database (IncytePharmaceuticals, Inc., Palo Alto, Calif.).

DESCRIPTION OF THE INVENTION

[0016] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0017] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0018] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0019] Definitions “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0020] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0021] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0022] HPAK, as used herein, refers to the amino acid sequences ofsubstantially purified HPAK obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0023] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0024] A “variant” of HPAK, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0025] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0026] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0027] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0028] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic HPAK, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0029] The term “agonist”, as used herein, refers to a molecule which,when bound to HPAK, causes a change in HPAK which modulates the activityof HPAK. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to HPAK.

[0030] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to HPAK, blocks or modulates the biologicalor immunological activity of HPAK. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to HPAK.

[0031] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of HPAK. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of HPAK.

[0032] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of HPAKor portions thereof and, as such, is able to effect some or all of theactions of kallikrein-like molecules.

[0033] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding HPAK or the encoded HPAK.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0034] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0035] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0036] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0037] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0038] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0039] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.”The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0040] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0041] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0042] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated.

[0043] The designation “negative” is sometimes used in reference to theantisense strand, and “positive” is sometimes used in reference to thesense strand.

[0044] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human HPAK and fragments thereof.

[0045] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0046] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0047] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0048] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding HPAKor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0049] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding HPAK in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0050] “Alterations” in the polynucleotide of SEQ ID NO: 2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding HPAK including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes HPAK (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HPAK (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0051] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind HPAKpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0052] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0053] The Invention

[0054] The invention is based on the discovery of a novel humankallikrein, (HPAK), the polynucleotides encoding HPAK, and the use ofthese compositions for the diagnosis, prevention, or treatment of cancerand disorders of the prostate.

[0055] Nucleic acids encoding the human HPAK of the present inventionwere first identified in Incyte Clone 964204 from the breast tissue cDNAlibrary (BRSTNOT05) through a computer-generated search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 964204 (BRSTNOT05), 875949 (LUNGAST01), 1325870 (LPARNOT02),1685649 (PROSTNOT15), and 1725220 (PROSNOT14).

[0056] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1Aand 1B. HPAK is 253 amino acids in length and has four potentialN-glycosylation sites at asparagine residues 102, 168, 184, and 213.HPAK has chemical and structural homology with human pancreatickallikrein (GI 186653; SEQ ID NO:3). In particular, HPAK and humanpancreatic kallikrein share 54% identity. HPAK's amino terminal 24 aminoacids are hydrophilic and closely resemble signal sequences importantfor kallikrein secretion (FIGS. 2, 3, and 4). HPAK sequence containsconserved residues critical for serine protease activity, H₆₅, D₁₁₃, andS₂₀₆ (FIG. 2). Amino acid residue D₂₀₀ is likely to confer on HPAKchymotrypsinogen-like activity. HPAK amino acid sequence includes 10conserved cysteine residues (31, 50, 66, 145, 166, 177, 191, 202, 212,and 227; FIG. 2). In the kallikreins mentioned above, these cysteinesare structurally important and form five disulfide bonds. As illustratedby FIGS. 3 and 4, HPAK and human pancreatic kallikrein have rathersimilar hydrophobicity plots. Northern analysis reveals the expressionpattern of this sequence in various libraries (FIG. 5). Of the 15tissues in which HPAK is expressed, six are from the prostate gland andseven come from cancer patients.

[0057] The invention also encompasses HPAK variants. A preferred HPAKvariant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the HPAK amino acid sequence (SEQ ID NO:1). Amost preferred HPAK variant is one having at least 95% amino acidsequence similarity to SEQ ID NO:1.

[0058] The invention also encompasses polynucleotides which encode HPAK.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of HPAK can be used to generate recombinant molecules whichexpress HPAK. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A and 1B.

[0059] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding HPAK, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring HPAK, and all such variations are to beconsidered as being specifically disclosed.

[0060] Although nucleotide sequences which encode HPAK and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HPAK under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HPAK or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HPAK and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0061] The invention also encompasses production of DNA sequences, orportions thereof, which encode HPAK and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding HPAK or any portionthereof.

[0062] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0063] Altered nucleic acid sequences encoding HPAK which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent HPAK. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HPAK. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of HPAK is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0064] Also included within the scope of the present invention arealleles of the genes encoding HPAK. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0065] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; M J Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0066] The nucleic acid sequences encoding HPAK may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0067] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0068] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0069] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0070] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0071] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. Genotyper™ andSequence Navigator™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0072] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode HPAK, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of HPAK in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressHPAK.

[0073] As will be understood by those of skill in the art, it may beadvantageous to produce HPAK-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0074] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterHPAK encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0075] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HPAK may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HPAK activity, it may be useful toencode a chimeric HPAK protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HPAK encoding sequence and theheterologous protein sequence, so that HPAK may be cleaved and purifiedaway from the heterologous moiety.

[0076] In another embodiment, sequences encoding HPAK may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HPAK, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

[0077] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of HPAK, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0078] In order to express a biologically active HPAK, the nucleotidesequences encoding HPAK or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0079] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding HPAKand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0080] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding HPAK. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0081] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript®) phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HPAK,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0082] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HPAK. For example, whenlarge quantities of HPAK are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding HPAK may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0083] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0084] In cases where plant expression vectors are used, the expressionof sequences encoding HPAK may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0085] An insect system may also be used to express HPAK. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding HPAKmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of HPAK will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which HPAK may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0086] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding HPAK may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing HPAK in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0087] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding HPAK. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding HPAK, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0088] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0089] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress HPAK may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0090] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0091] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding HPAK isinserted within a marker gene sequence, recombinant cells containingsequences encoding HPAK can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HPAK under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0092] Alternatively, host cells which contain the nucleic acid sequenceencoding HPAK and express HPAK may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0093] The presence of polynucleotide sequences encoding HPAK can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding HPAK.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding HPAK todetect transformants containing DNA or RNA encoding HPAK. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0094] A variety of protocols for detecting and measuring the expressionof HPAK, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HPAK is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.

[0095] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HPAKinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding HPAK, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0096] Host cells transformed with nucleotide sequences encoding HPAKmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode HPAK may be designed to contain signal sequences which directsecretion of HPAK through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingHPAK to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and HPAK may be used to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincontaining HPAK and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site.

[0097] The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinasecleavage site provides a means for purifying HPAK from the fusionprotein. A discussion of vectors which contain fusion proteins isprovided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0098] In addition to recombinant production, fragments of HPAK may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431 APeptide Synthesizer (Perkin Elmer). Various fragments of HPAK may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0099] Therapeutics

[0100] Chemical and structural homology exists among HPAK, humanpancreatic kallikrein, and african rat renal kallikrein. In addition,northern analysis (FIG. 5) shows that six cDNA libraries containing HPAKtranscripts are from prostate tissue, HPAK is believed to function inthe prostate gland. Of the six prostate tissue libraries, five were frompatients with prostate cancer. HPAK expression was also found in breastand parotid gland tissues from patients with tumors in these tissues.Thus, HPAK expression appears to be associated with the prostate glandand in the development of cancer.

[0101] Antagonists or inhibitors of HPAK may be used to suppressexcessive cell proliferation. Thus in one embodiment, antagonists orinhibitors of HPAK may be administered to a subject to treat or preventcancer, including but not limited to, cancer of the prostate, parotidgland, and breast.

[0102] More specifically, antagonists or inhibitors of HPAK may be usedto suppress excessive proliferation of prostate cells.

[0103] In other aspects, antibodies which are specific for HPAK may beused directly as an antagonist, or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress HPAK.

[0104] In another embodiment, a vector expressing antisense of thepolynucleotide encoding HPAK may be administered to a subject to treator prevent cancer. Examples of cancers include, but are not limited to,cancer of the breast and parotid gland.

[0105] In another embodiment, a vector expressing antisense of thepolynucleotide encoding HPAK may be administered to a subject to treator prevent disorders of the prostate. Examples of such disorders of theprostate include, but are not limited to, prostate cancer and benignprostatic hyperplasia.

[0106] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0107] Antagonists or inhibitors of HPAK may be produced using methodswhich are generally known in the art. In particular, purified HPAK maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind HPAK.

[0108] The antibodies may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0109] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith HPAK or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0110] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to HPAK have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of HPAK amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0111] Monoclonal antibodies to HPAK may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0112] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHPAK-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0113] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0114] Antibody fragments which contain specific binding sites for HPAKmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0115] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between HPAK and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HPAK epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0116] In another embodiment of the invention, the polynucleotidesencoding HPAK, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding HPAK may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding HPAK. Thus, antisense molecules may be used to modulate HPAKactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding HPAK.

[0117] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingHPAK. These techniques are described both in Sambrook et al. (supra) andin Ausubel et al. (supra).

[0118] Genes encoding HPAK can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes HPAK. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0119] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding HPAK, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0120] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding HPAK.

[0121] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0122] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding HPAK. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0123] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0124] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0125] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0126] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HPAK,antibodies to HPAK, mimetics, agonists, antagonists, or inhibitors ofHPAK. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0127] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0128] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0129] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0130] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0131] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0132] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0133] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0134] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0135] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0136] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0137] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HPAK, such labeling wouldinclude amount, frequency, and method of administration.

[0138] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0139] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0140] A therapeutically effective dose refers to that amount of activeingredient, for example HPAK or fragments thereof, antibodies of HPAK,agonists, antagonists or inhibitors of HPAK, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0141] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0142] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0143] Diagnostics

[0144] In another embodiment, antibodies which specifically bind HPAKmay be used for the diagnosis of conditions or diseases characterized byexpression of HPAK, or in assays to monitor patients being treated withHPAK, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for HPAK includemethods which utilize the antibody and a label to detect HPAK in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0145] A variety of protocols including ELISA, RIA, and FACS formeasuring HPAK are known in the art and provide a basis for diagnosingaltered or abnormal levels of HPAK expression. Normal or standard valuesfor HPAK expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HPAK under conditions suitable for complex formation Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of HPAKexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0146] In another embodiment of the invention, the polynucleotidesencoding HPAK may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHPAK may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HPAK,and to monitor regulation of HPAK levels during therapeuticintervention.

[0147] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HPAK or closely related molecules, may be used to identifynucleic acid sequences which encode HPAK. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding HPAK, alleles, or related sequences.

[0148] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the HPAK encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring HPAK.

[0149] Means for producing specific hybridization probes for DNAsencoding HPAK include the cloning of nucleic acid sequences encodingHPAK or HPAK derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0150] Polynucleotide sequences encoding HPAK may be used for thediagnosis of conditions or diseases which are associated with expressionof HPAK. Examples of such conditions or diseases include pancreaticdisorders, asthma, and cancers of the prostate, parotid gland, andbreast. The polynucleotide sequences encoding HPAK may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pIN, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered HPAK expression. Such qualitative or quantitative methods arewell known in the art.

[0151] In a particular aspect, the nucleotide sequences encoding HPAKmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding HPAK may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding HPAK in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0152] In order to provide a basis for the diagnosis of diseaseassociated with expression of HPAK, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes HPAK, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0153] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0154] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0155] Additional diagnostic uses for oligonucleotides designed from thesequences encoding HPAK may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′->3′) and another withantisense (3′<-5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0156] Methods which may also be used to quantitate the expression ofHPAK include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0157] In another embodiment of the invention, the nucleic acidsequences which encode HPAK may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0158] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981 f). Correlation between the location of thegene encoding HPAK on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0159] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0160] In another embodiment of the invention, HPAK, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenHPAK and the agent being tested, may be measured.

[0161] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to HPAK large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with HPAK, or fragments thereof, and washed. Bound HPAK is thendetected by methods well known in the art. Purified HPAK can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0162] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding HPAKspecifically compete with a test compound for binding HPAK. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HPAK.

[0163] In additional embodiments, the nucleotide sequences which encodeHPAK may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0164] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0165] I BRSTNOT05 cDNA Library Construction

[0166] The breast (BRSTNOT05) cDNA library was constructed from thebreast tissue of a 58 year old Caucasian female. The patient wasdiagnosed with multicentric invasive grade 4 lobular carcinoma.

[0167] The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.0,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water and DNase treated at 37° C. Extractionand precipitation were repeated. The mRNA then isolated with the QiagenOligotex kit (QIAGEN Inc; Chatsworth Calif.) and used to construct thecDNA library.

[0168] The mRNA was handled according to the recommended protocols inthe SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Cat. #18248-013; Gibco/BRL, Gaithersburg, Md.), cDNAs were fractionatedon a Sepharose CL4B column (Cat. #275105-01; Pharmacia), and those cDNAsexceeding 400 bp were ligated into pSport I. The plasmid pSport I wassubsequently transformed into DH5a™ competent cells (Cat. #18258-012;Gibco/BRL).

[0169] II Isolation and Sequencing of cDNA Clones

[0170] Plasmid DNA was released from the cells and purified using theREAL Prep 96 Plasmid Kit (Catalog #26173; QIAGEN, Inc). This kit enablesthe simultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, LIFE TECHNOLOGIES™,Gaithersburg, Md.) with carbenicillin at 25 mg/L and glycerol at 0.4%;2) after inoculation, the cultures were incubated for 19 hours and atthe end of incubation, the cells were lysed with 0.3 ml of lysis buffer;and 3) following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4°C.

[0171] The cDNAs were sequenced by the method of Sanger F. and A. R.Coulson (1975; J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with Peltier Thermal Cyclers(PTC200 from M J Research, Watertown Mass.) and Applied Biosystems 377DNA Sequencing Systems; and the reading frame was determined.

[0172] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0173] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul (1993) supra,Altschul (1990) supra).

[0174] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R. F. and T. F. Smith(1992 Protein Engineering 5:35-51), incorporated herein by reference,can be used when dealing with primary sequence patterns and secondarystructure gap penalties. As disclosed in this application, the sequenceshave lengths of at least 49 nucleotides, and no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

[0175] The BLAST approach, as detailed in Karlin and Altschul (supra)and incorporated herein by reference, searches for matches between aquery sequence and a database sequence, to evaluate the statisticalsignificance of any matches found, and to report only those matcheswhich satisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

[0176] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology. The relevantdatabase for a particular match were reported as a GIxxx+p (where xxx ispri, rod, etc and if present, p=peptide). The product score iscalculated as follows: the % nucleotide or amino acid identity [betweenthe query and reference sequences] in BLAST is multiplied by the %maximum possible BLAST score [based on the lengths of query andreference sequences] and then divided by 100. Where an Incyte Clone washomologous to several sequences, up to five matches were provided withtheir relevant scores. In an analogy to the hybridization proceduresused in the laboratory, the electronic stringency for an exact match wasset at 70, and the conservative lower limit for an exact match was setat approximately 40 (with 1-2% error due to uncalled bases).

[0177] IV Northern Analysis

[0178] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0179] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0180] The basis of the search is the product score which is defined as:$\frac{{\% \quad {sequence}\quad {identity} \times \quad \% {\quad \quad}{maximum}\quad {BLAST}\quad {score}}\quad}{\quad 100}$

[0181] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0182] The results of northern analysis are reported as a list oflibraries in which the transcript encoding HPAK occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0183] V Extension of HPAK-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

[0184] Full length HPAK-encoding nucleic acid sequence (SEQ ID NO:2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0185] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0186] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (and holding)

[0187] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0188] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0189] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0190] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0191] VI Labeling and Use of Hybridization Probes

[0192] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

[0193] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0194] VII Antisense Molecules

[0195] Antisense molecules to the HPAK-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring HPAK. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of HPAK, as shown in FIGS. 1A and 1B, is used toinhibit expression of naturally occurring HPAK. The complementaryoligonucleotide is designed from the most unique 5′ sequence as shown inFIGS. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an HPAK-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5′ sequence of SEQ IDNO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal or 5′coding sequence of the polypeptide as shown in FIGS. 1A and 1B.

[0196] VIII Expression of HPAK

[0197] Expression of HPAK is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express HPAK in E. coli.Upstream of the cloning site, this vector contains a promoter forB-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0198] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of HPAK into the bacterial growth media which can be useddirectly in the following assay for activity.

[0199] IX Demonstration of HPAK Activity

[0200] HPAK's proteolytic activity can be determined by methodsdescribed by Christernsson A. et al. (1990, Eur. J. Biochem. 194:755-763). Substrates for proteolytic cleavage are found in human semen.Human seminal plasma is collected and coagulated semen is washed free ofsoluble components. HPAK is incubated with coagulated semen in a bufferconsisting of 50 mmol/I TRIS-HCl pH 7.8, with 0.1 mol/l NaCl. Reactionsare performed at 37° C. After incubation periods of different durations(from 0 to 30 minutes) the samples are analyzed by SDS/PAGE. Theresulting pattern of peptide fragments is compared and quantitated usinga control sample in which HPAK is not added.

[0201] X Production of HPAK Specific Antibodies

[0202] HPAK that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0203] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0204] XI Purification of Naturally Occurring HPAK Using SpecificAntibodies

[0205] Naturally occurring or recombinant HPAK is substantially purifiedby immunoaffinity chromatography using antibodies specific for HPAK. Animmunoaffinity column is constructed by covalently coupling HPAKantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0206] Media containing HPAK is passed over the immunoafinity column,and the column is washed under conditions that allow the preferentialabsorbance of HPAK (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HPAK binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and HPAK is collected.

[0207] XII Identification of Molecules Which Interact with HPAK

[0208] HPAK or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HPAK, washed and any wells withlabeled HPAK complex are assayed. Data obtained using differentconcentrations of HPAK are used to calculate values for the number,affinity, and association of HPAK with the candidate molecules.

[0209] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 262 amino acids amino acid single linear 1 Met Trp Phe Leu Val LeuCys Leu Ala Leu Ser Leu Gly Gly Thr Gly 1 5 10 15 Ala Ala Pro Pro IleGln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Gln His Ser Gln ProTrp Gln Ala Ala Leu Tyr His Phe Ser Thr Phe 35 40 45 Gln Cys Gly Gly IleLeu Val His Arg Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys Ile Ser AspAsn Tyr Gln Leu Trp Leu Gly Arg His Asn Leu 65 70 75 80 Phe Asp Asp GluAsn Thr Ala Gln Phe Val His Val Ser Glu Ser Phe 85 90 95 Pro His Pro GlyPhe Asn Met Ser Leu Leu Glu Asn His Thr Arg Gln 100 105 110 Ala Asp GluAsp Tyr Ser His Asp Leu Met Leu Leu Arg Leu Thr Glu 115 120 125 Pro AlaAsp Thr Ile Thr Asp Ala Val Lys Val Val Glu Leu Pro Thr 130 135 140 GlnGlu Pro Glu Val Gly Ser Thr Cys Leu Ala Ser Gly Trp Gly Ser 145 150 155160 Ile Glu Pro Glu Asn Phe Ser Phe Pro Asp Asp Leu Gln Cys Val Asp 165170 175 Leu Lys Ile Leu Pro Asn Asp Glu Cys Glu Lys Ala His Val Gln Lys180 185 190 Val Thr Asp Phe Met Leu Cys Val Gly His Leu Glu Gly Gly LysAsp 195 200 205 Thr Cys Val Gly Asp Ser Gly Gly Pro Leu Met Cys Asp GlyVal Leu 210 215 220 Gln Gly Val Thr Ser Trp Gly Tyr Val Pro Cys Gly ThrPro Asn Lys 225 230 235 240 Pro Ser Val Ala Val Arg Val Leu Ser Tyr ValLys Trp Ile Glu Asp 245 250 255 Thr Ile Ala Glu Asn Ser 260 833 basepairs nucleic acid single linear 2 GCTGGCCCCT GGACACCTCT GTCACCATGTGGTTCCTGGT TCTGTGCCTC GCCCTGTCCC 60 TGGGGGGGAC TGGTGCTGCG CCCCCGATTCAGTCCCGGAT TGTGGGAGGC TGGGAGTGTG 120 AGCAGCATTC CCAGCCCTGG CAGGCGGCTCTGTACCAGAA GACGCGGCTA CTCTGTGGGG 180 CGACGNTCAT NGCCCCCAGA TGGTTCCTGACAGCAGCCCA CTGCCTNAAG CCCCGCTACA 240 TAGTTCACCT GGGGCAGCAC AACCTCCAGAAGGAGGAGGG CTGTGAGCAG ACCCGGACAG 300 CCACTGAGTC CTTCCCCCAC CCCGGCTTCAACAACAGCCT CCCCAACAAA GACCACCGCA 360 ATGACATCAT GCTGGTGAAG ATGGCATCGCCAGTCTCCAT CACCTGGGCT GTGCGACCCC 420 TCACCCTCTC CTCACGCTGT GTCACTGCTGGCACCAGCTG CCTCATTTCC GGCTGGGGCA 480 GCACGTCCAG CCCCCAGTTA CGCCTGCCTCACACCTTGCG ATGCGCCAAC ATCACCATCA 540 TTGAGCACCA GAAGTGTGAG AACGCCTACCCCGGCAACAT CACAGACACC ATGGTGTGTG 600 CCAGCGTGCA GGAAGGGGGC AAGGACTCCTGCCAGGGTGA CTCCGGGGGC CCTCTGGTCT 660 GTAACCAGTC TCTTCAAGGC ATTATCTCCTGGGGCCAGGA TCCGTGTGCG ATCACCCGAA 720 AGCCTGGTGT CTACACGAAA GTCTGCAAATATGTGGACTG GATCCAGGAG ACGATGAAGA 780 ACAATTAGAC TGGACNTCAC CTCCGAANCCCCCACAGCCC ATCACCCTCC ATT 833 262 amino acids amino acid single linearGenBank 186653 3 Met Trp Phe Leu Val Leu Cys Leu Ala Leu Ser Leu Gly GlyThr Gly 1 5 10 15 Ala Ala Pro Pro Ile Gln Ser Arg Ile Val Gly Gly TrpGlu Cys Glu 20 25 30 Gln His Ser Gln Pro Trp Gln Ala Ala Leu Tyr His PheSer Thr Phe 35 40 45 Gln Cys Gly Gly Ile Leu Val His Arg Gln Trp Val LeuThr Ala Ala 50 55 60 His Cys Ile Ser Asp Asn Tyr Gln Leu Trp Leu Gly ArgHis Asn Leu 65 70 75 80 Phe Asp Asp Glu Asn Thr Ala Gln Phe Val His ValSer Glu Ser Phe 85 90 95 Pro His Pro Gly Phe Asn Met Ser Leu Leu Glu AsnHis Thr Arg Gln 100 105 110 Ala Asp Glu Asp Tyr Ser His Asp Leu Met LeuLeu Arg Leu Thr Glu 115 120 125 Pro Ala Asp Thr Ile Thr Asp Ala Val LysVal Val Glu Leu Pro Thr 130 135 140 Gln Glu Pro Glu Val Gly Ser Thr CysLeu Ala Ser Gly Trp Gly Ser 145 150 155 160 Ile Glu Pro Glu Asn Phe SerPhe Pro Asp Asp Leu Gln Cys Val Asp 165 170 175 Leu Lys Ile Leu Pro AsnAsp Glu Cys Glu Lys Ala His Val Gln Lys 180 185 190 Val Thr Asp Phe MetLeu Cys Val Gly His Leu Glu Gly Gly Lys Asp 195 200 205 Thr Cys Val GlyAsp Ser Gly Gly Pro Leu Met Cys Asp Gly Val Leu 210 215 220 Gln Gly ValThr Ser Trp Gly Tyr Val Pro Cys Gly Thr Pro Asn Lys 225 230 235 240 ProSer Val Ala Val Arg Val Leu Ser Tyr Val Lys Trp Ile Glu Asp 245 250 255Thr Ile Ala Glu Asn Ser 260 263 amino acids amino acid single linearGenBank 55527 4 Met Trp Phe Leu Ile Leu Phe Leu Ala Leu Phe Leu Gly GlyIle Asp 1 5 10 15 Ala Ala Pro Pro Val Gln Ser Arg Ile Ile Gly Gly PheAsn Cys Glu 20 25 30 Lys Asn Ser Gln Pro Trp His Val Ala Val Tyr Arg PheAla Arg Tyr 35 40 45 Gln Cys Gly Gly Val Leu Leu Asp Ala Asn Trp Val LeuThr Ala Ala 50 55 60 His Cys Tyr Asn Asp Lys Tyr Gln Val Trp Leu Gly LysAsn Asn Arg 65 70 75 80 Phe Glu Asp Glu Pro Ser Ala Gln His Gln Leu IleSer Lys Ala Ile 85 90 95 Pro His Pro Gly Phe Asn Met Ser Leu Leu Asn LysAsp His Thr Pro 100 105 110 His Pro Glu Asp Asp Tyr Ser Asn Asp Leu MetLeu Val Arg Leu Lys 115 120 125 Lys Pro Ala Glu Ile Thr Asp Val Val LysPro Ile Asp Leu Pro Thr 130 135 140 Glu Glu Pro Thr Val Gly Ser Arg CysLeu Ala Ser Gly Trp Gly Ser 145 150 155 160 Thr Thr Pro Thr Glu Glu PheGlu Tyr Ser His Asp Leu Gln Cys Val 165 170 175 Tyr Leu Glu Leu Leu SerAsn Glu Val Cys Ala Lys Ala His Thr Glu 180 185 190 Lys Val Thr Asp ThrMet Leu Cys Ala Gly Glu Met Asp Gly Gly Lys 195 200 205 Asp Thr Cys ValGly Asp Ser Gly Gly Pro Leu Ile Cys Asp Gly Val 210 215 220 Leu Gln GlyIle Thr Ser Trp Gly Pro Thr Pro Cys Ala Leu Pro Asn 225 230 235 240 ValPro Gly Ile Tyr Thr Lys Leu Ile Glu Tyr Arg Ser Trp Ile Lys 245 250 255Asp Val Met Ala Asn Asn Pro 260

What is claimed is:
 1. A substantially purified prostate-associatedkallikrein protein comprising the amino acid sequence of SEQ ID NO:1 orfragments thereof.
 2. An isolated and purified polynucleotide sequenceencoding the prostate-associated kallikrein protein of claim
 1. 3. Apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of claim
 2. 4. A hybridization probecomprising the polynucleotide sequence of claim
 2. 5. An isolated andpurified polynucleotide sequence comprising SEQ ID NO:2 or variantsthereof.
 6. A polynucleotide sequence which is complementary to thepolynucleotide sequence of claim 2 or variants thereof.
 7. Ahybridization probe comprising the polynucleotide sequence of claim 6.8. An expression vector containing the polynucleotide sequence of claim2.
 9. A host cell containing the vector of claim
 8. 10. A method forproducing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 the method comprising the steps of: a) culturing the host cell ofclaim 9 under conditions suitable for the expression of the polypeptide;and b) recovering the polypeptide from the host cell culture.
 11. Apurified antibody which binds specifically to the polypeptide ofclaim
 1. 12. A purified agonist which specifically binds to andmodulates the activity of the polypeptide of claim
 1. 13. A purifiedantagonist which specifically binds to and modulates the activity of thepolypeptide of claim
 1. 14. A pharmaceutical composition comprising asubstantially purified antagonist of claim 13 in conjunction with asuitable pharmaceutical carrier.
 15. A method for treating cancercomprising administering to a subject in need of such treatment aneffective amount of the pharmaceutical composition of claim
 14. 16. Amethod for treating disorders of the prostate comprising administeringto a subject in need of such treatment an effective amount of thepharmaceutical composition of claim
 14. 17. A method for detection ofpolynucleotides encoding kallikrein in a biological sample comprisingthe steps of: a) hybridizing the polynucleotide of claim 6 to nucleicacid material of a biological sample, thereby forming a hybridizationcomplex; and b) detecting said hybridization complex, wherein thepresence of said complex correlates with the presence of apolynucleotide encoding kallikrein in said biological sample.